The production of haploid plants generated through either anther or isolated microspore culture has succeeded in over 240 species from 85 genera in 38 families (Srivastava and Johri 1988). Microspore culture of Brassica napus has become one of the most efficient embryogenic systems and has been exploited for developmental studies (e.g. Zaki and Dickinson 1991; Telmer et al. 1992, 1993, 1994), for mutagenesis and gene transfer (Swanson et al. 1989; Huang 1992), and for development of doubled haploid homozygous breeding lines (Chen and Beversdorf 1992). The use of haploid plants, generated from anther or microspore culture, has enhanced the efficiency of crop improvement programs (Collins and Genovesi 1982, Chen and Beversdorf 1992). Although haploid plants can be readily regenerated, the haploids cannot be used directly in genetic studies and breeding programs because they are sterile (Subrahmanyam and Kasha 1975). The current methods of doubling the chromosome complement of haploids to produce fertile homozygous doubled haploids are inefficient and labor intensive.
Efficient induction of embryogenesis is necessary for developmental and biochemical studies. The efficiency of embryogenesis of B. napus has been improved by using donor plants grown at low temperatures (Keller et al. 1986), by optimizing the microspore culturing conditions (Keller et al. 1986; Lichter 1981; Fan et al. 1988; Chuong and Beaversdorf 1985; Kott et al. 1988; Gland et al. 1988; Huang et al. 1990) and by using microspores at the competent developmental stages (Telmer et al. 1992).
Exposure of microspores to a high temperature (32.5.degree. C.) is considered to be a key factor for induction of embryogenesis (Keller and Armstrong 1978; Cordewener et al. 1994) and it has been proposed that heat shock proteins play a role in the inductive process (Pechan et al. 1992). Several unique proteins, synthesized during heat induction, have been identified and it has been suggested that they may be early markers of embryogenesis or heat shock proteins involved in the induction process (Cordewener et al. 1994). However, with the use of heat shock to induce embryogenesis, it is very difficult to distinguish between factors associated with the heat shock process and those specific to the embryogenic process. Attempts to replace heat induction with alternate methods such as gamma irradiation or ethanol treatments resulted in very low embryo induction (Pechan and Keller 1989). However, the induction of sporophytic development, by means other than heat, would be very valuable to allow discrimination of heat shock factors and embryogenic factors and thereby identify the critical events involved in the change from gametophytic to sporophytic development.
Microspore morphology is altered by the 32.5.degree. C. heat treatment. Although several morphological changes have been identified in B. napus cv. Topas, including the appearance of cytoplasmic granules and organelle-free regions, plasma membrane associated electron-dense deposits, and microtubule reorganization, the most prominent change is the dislocation of the nucleus (Fan et al. 1988; Simmonds et al. 1991; Telmer et al. 1993, 1994; Simmonds 1994). During pollen ontogenesis the nucleus of an early and mid-unicellular (MU) microspore is centrally located; during vacuolar enlargement, it is relocated to a lateral position, the unicellular-vacuolate stage (UV); and it remains appressed to the edge of the cell in the late-unicellular (LU) stage after the disappearance of the large vacuole (Telmer et al. 1992, 1993). The LU microspore enters the first pollen mitosis which is acentric and results in an asymmetrical division comprising a small generative cell and a large vegetative cell separated by an unstable cell wall (Telmer et al. 1993). If the LU microspore is subjected to the heat treatment, the nucleus migrates to a more central position where mitosis occurs and ultimately results in a symmetrical division with two daughter cells similar in size and organelle distribution, and separated by a stable cell wall (Fan et al. 1988; Telmer et al 1993; Simmonds 1994); the symmetric division blocks further pollen development and identifies the induced structures (Telmer et al. 1994). An early structural marker which predicts a change in microspore division symmetry is a preprophase band (PPB) of microtubules; the PPB, a cortical ring of microtubules, appears in the medial region of the microspore after only about 6-8 h of heat treatment (Simmonds et al. 1991; Simmonds 1994). PPBs have not been observed during pollen development (Van Lammeren et al. 1985; Terasaka and Niitsu 1990; Simmonds et al. 1991). As the PPBs predict the position of the future division plane in organized (Gunning and Hardham 1982) and disorganized tissue (Simmonds 1986), and may have a role in wall stabilization (Mineyuki and Gunning 1990), it has been proposed that microtubule reorganization is a key event in changing developmental patterns where altered division symmetry and cell wall dynamics define the induced embryogenic structure (Simmonds 1994).
Spontaneous diploids have been reported to arise from anther culture of barley (Subrahmanyam and Kasha 1975), tobacco (Burk et al. 1972, Kasperbauer and Collins 1972;), corm (Ku et al. 1981) and B. napus (Charne et al. 1988). It has been hypothesized that diploids may occur through endomitosis, endoreduplication and/or nuclear fusion within the cell during early stages of culture (Sunderland et al. 1974, Keller and Armstrong 1978) and possibly from unreduced gametes (Wenzel et al. 1977, Chen and Beversdorf 1992). However, as the occurrence of spontaneous diploids is an infrequent and inconsistent event, colchicine has been used to increase the frequency. The techniques of colchicine application has not changed much since Levan (1938) soaked onion roots in colchicine solutions. Currently, apical meristems, secondary buds, tillers or roots are treated with colchicine (see Wong 1989, Swanson 1990, Mathias and Robbelen 1991). Generally, about 50% of the treated plants are responsive. These procedures are labor intensive (Chen and Beversdorf 1992), hazardous (Depaepe et al. 1981, Hansen et al. 1988, Barnabas et al. 1991, Hassawi and Liang 1991) and costly (Hassawi and Liang 1991) because high concentrations of colchicine are needed. Furthermore, three months can be added to the plant regeneration time to recover homozygous lines (Beversdorf et al. 1987). Additional drawbacks to using this approach include the regeneration of chimeras (Hansen et al. 1988, Wan et al. 1989, Wong 1989, Swanson 1990, Bamabas et al. 1991), aneuploids (Zhao and Davidson 1984), abnormalities in plant development (Hart and Sabnis 1976, Loh and Ingram 1983) and low seed yield. Application of colchicine to cultures prior to organ formation has produced non-chimeric doubled haploids from corn callus (Wan et al. 1989) and wheat anther culture (Barnabas et al. 1991). An effective alternative to colchicine has not been reported to date but would be highly desirable (Wan et al. 1989, Hassawi and Liang 1991).
Trifluralin, a dinitroaniline herbicide (Probst et al. 1976), acts in a manner similar to colchicine, by disrupting spindle microtubules (Bartels and Hilton 1973). Trifluralin, unlike colchicine, has a higher affinity for plant tubulin than for animal tubulin (Hess and Bayer 1977, Morejohn and Fosket 1984, Morejohn et al. 1984).
Non-chimeric doubled haploid plants were recovered from B. napus cv. Topas microspores cultured in the presence of colchicine or trifluralin, according to the present invention. These antimitotic agents were applied during the initial stages of culturing, while the microspores were undergoing the heat treatment (32.5.degree. C.) used to induce embryogenesis. Trifluralin treated cultures generated normal embryos which germinated directly upon transfer to regeneration medium and produced doubled haploid plants at frequencies approaching 60%. However, only about 20% of the plants recovered from colchicine treated cultures were doubled haploids. Longer colchicine treatments resulted in higher frequencies of fertile plants but embryo development was abnormal and several subcultures were required to induce plant development. Chen et al. (1994) also found that the cv. Topas responded negatively to colchicine treatment, however other cultivars responded positively by increasing the production of embryos and the frequency of fertile plants. However, it has been shown that colchicine can be used, instead of heat, to induce embryogenesis from B. napus cv. Topas microspores. The embryos generated from colchicine-induced cultures, at non-inductive temperatures were normal. Ninety percent of the plants recovered from these embryos were fertile.
Desirable genetic recombinants resulting from microsporogenesis can be exploited by recovering haploid plants from microspore derived embryos. The interesting recombinants can be used for the development of new varieties or homozygous breeding lines. As haploid plants are sterile, the practical utilization of haploids in breeding programs relies on an efficient chromosome doubling technique to obtain fertile diploid plants (Subrahmanyam and Kasha 1975, Loh and Ingram 1983).